Polypeptide eye absorption enhancer and use thereof

a polypeptide and eye absorption technology, applied in the field of pharmaceutical preparations, can solve the problems of poor absorption effect, short residence time of ordinary eye drops in the conjunctival sac, and low bioavailability of eye drops, so as to improve the bioavailability of eye chemicals, promote drug absorption in the eye, and reduce compliance

Active Publication Date: 2020-07-30
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0023]The penetratin derivatives described in the present invention form a covalent complex with a drug having a diagnostic or therapeutic effect, or modified on the surface of drug delivery systems such as liposomes, micelles, and nanoparticles that contain diagnostic or therapeutic drugs, or self-assemble with bio-macromolecules drugs such as genes, polypeptides and proteins with negative charges under physiological conditions to form non-covalent nanocomplexes, after intraocular administration through the conjunctival sac, it could promote the drug to pass through many ocular absorption barriers (cornea, conjunctiva, sclera, etc.) into the eye, even the chemical drugs or biomacromolecules drugs such as genes, polypeptides, and protein they carry are delivered to the retina in the posterior segment of the eye. In the covalent complex, surface-modified nano drug delivery system, or non-covalent nanocomplex constructed using the penetratin derivative, the concentration of the penetratin derivative is 1 nM-500 μM, preferably 10 nM-300 μM, and more preferably 100 nM-100 μM. This intraocular drug delivery system administered through a non-traumatic route helps to promote drug absorption in the eye, improve the eye bioavailability of chemical drugs and biomacromolecules, and could be used clinically as an alternative to intraocular injection and other administration with low compliance.
[0024]In order to show the ocular absorption promotion effect of the penetratin derivatives described in the present invention on a chemical drug covalently connected thereto, taking a tryptophan (W)-monosubstituted penetratin derivative as an example, one additional lysine (K) was attached to the C-terminus of the tryptophan-substituted penetratin derivative and a fluorescent probe carboxyfluorescein (FAM) was linked to the amino side chain of lysine to form a covalent complex. Through a series of in vitro and in vivo experiments, the present invention investigated the ocular cell uptake ability of penetratin derivative-modified fluorescent probes, permeability of ocular tissues in vitro and absorption and distribution in the eye of living animals after intraocular administration through intraconjunctival sac. The results show that compared with the wild-type penetratin, the tryptophan-substituted penetratin derivative has significantly enhanced ocular absorption promotion ability.
[0025]More importantly, the applicant found that the penetrating ability of penetratin derivatives to ocular tissue is related to its hydrophobicity (lipophilicity). The stronger the hydrophobicity (lipophilicity), the stronger penetrating ability of penetratin derivatives to ocular tissue. Therefore, penetratin derivatives prepared using other hydrophobic amino acids would also achieve enhanced ocular absorption promotion effects. Moreover, the applicant confirmed that penetratin derivatives could effectively deliver fluorescent probes into the eye, and if fluorescent probes were replaced with other diagnostic or therapeutic drugs, the same intraocular delivery effect would be achieved.
[0026]In order to show the eye absorption promotion effect of penetratin derivative in the present invention on nano-drug carrier and bio-macromolecule drug through non-covalent binding thereto, taking a phenylalanine (F)-monosubstituted penetratin derivative as an example, and using antisense oligonucleotides as biological macromolecular drug models, a non-covalent complex containing antisense oligonucleotide was constructed in the presence of polyamide-amine dendrimer (PAMAM) and hyaluronic acid (HA). Through a series of in vitro and in vivo experiments, the present invention investigated the ocular cell uptake ability of penetratin derivative-modified non-covalent complexes, permeability of ocular tissues in vitro and absorption and distribution in the eye of living animals after intraocular administration through intraconjunctival sac. The results show that compared with the wild-type penetratin, the phenylalanine substituted penetratin derivative has significantly enhanced ocular absorption promotion ability.
[0027]The advantages of the penetratin derivatives in the present invention are that compared to small molecule absorption enhancers that are rarely used in the eye due to safety issues, such polypeptide absorption enhancers are easy to degrade and thus have better biological safety. On the other hand, polypeptide absorption enhancers are easy to be modified to achieve different application goals, and the penetratin derivatives of the present invention have stronger ocular absorption promotion capabilities than wild-type penetratin of natural origin.

Problems solved by technology

Ordinary eye drops have a short residence time in the conjunctival sac and have a poor absorption effect.
In particular, biological macromolecular drugs such as genes, peptides, and proteins are administered through local eye drops, and the bioavailability of the eye is extremely low, and almost could not reach the posterior segment of the eyes.
Intraocular injections and intraocular implants have high bioavailability, but patients have poor compliance and are likely to cause serious complications.

Method used

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  • Polypeptide eye absorption enhancer and use thereof
  • Polypeptide eye absorption enhancer and use thereof
  • Polypeptide eye absorption enhancer and use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0045]Preparation of covalently linked complexes of Penetratin derivatives and small molecular substances: based on the structure of wild-type penetratin, the sequences of the original basic amino acid and hydrophobic amino acid were kept unchanged, then peptide solid-phase synthesis technology was used to replace hydrophilic amino acids glutamine (Q) and asparagine (N) in penetratin molecules with hydrophobic tryptophan (W), respectively, thereby a series of polypeptide derivatives were obtained. One additional lysine (K) was attached to the C-terminus of the tryptophan-substituted penetratin derivative, and a fluorescent probe carboxyfluorescein (FAM) was linked to the amino side chain of lysine to form a covalent complex, the amino acid sequence of the covalent complex is shown in Table 2.

[0046]At the same time, the sequences of the original basic amino acid and hydrophilic amino acid the wild-type penetratin were kept unchanged, then peptide solid-phase synthesis technology was ...

example 2

[0049]Qualitative evaluation of cell uptake of the covalently linked complex of Penetratin derivatives and small molecular substances: well-growing human corneal epithelial cells (HCEC) and human conjunctival epithelial cells (NHC) were inoculated into 24-well plates at 5×103 cells / cm2, respectively. In the well plate, the culture solution was changed once a day after inoculation, and the experiment was performed after 2 to 3 days of culture. After discarding the culture solution, washing it three times with sterile PBS, adding serum-free DMEM solution containing 100 nM complex of penetratin derivative and FAM, and incubating at 37° C. and 5% CO2 for a period of time (0.5 h, 1 h, 2 h and 4 h). After the treatment, the solution was discarded, and the positively charged adsorbed substance was washed away with a PBS buffer solution containing 0.02 mg / mL heparin sodium, observed under inverted fluorescence microscope after staining cell nuclei with diimidylphenylindole (DAPI).

[0050]The ...

example 3

[0051]Quantitative evaluation of cell uptake of the covalently linked complex of Penetratin derivatives and small molecular substances: well-growing HCEC and NHC cells were inoculated into 24-well plates at 5×103 cells / cm2, respectively. In the well plate, the culture solution was changed once a day after inoculation, and the experiment was performed after 2 to 3 days of culture. After discarding the culture solution, washing it three times with sterile PBS, adding serum-free DMEM solution containing 3 μM complexes of penetratin derivative and FAM, and incubating at 37° C. and 5% CO2 for 4 h. When finished, the solution was discarded, and the positively charged adsorbed substance was washed away with a PBS buffer solution containing 0.02 mg / mL heparin sodium, the cells were digested, resuspended in 200 μL of sterile PBS buffer solution, and flow cytometry was performed after pipetting. The cell count of each sample was 104. Unadministered cells served as a negative control group.

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Abstract

The present disclosure belongs to the field of pharmaceutical preparations and relates to the design of a series of lipophilic derivatives by using wild-type penetrating peptide penetratin. These penetratin derivatives have a strong ability to penetrate the ocular tissues and do not cause ocular tissue toxicity. As ocular absorption enhancers, non-invasive routes could be used to achieve intraocular drug delivery and increase the ocular bioavailability of drugs. These penetratin derivatives and the ophthalmic drug delivery system constructed by them are used for eye drop administration, which could replace the intraocular injection with poor patients compliance, which greatly enhances the convenience and safety of the treatment of intraocular and fundus diseases.

Description

FIELD OF THE INVENTION[0001]The present invention belongs to the field of pharmaceutical preparations, and relates to the design of a series of lipophilic derivatives by wild-type penetrating peptide penetratin. These penetratin derivatives have very strong ability to penetrate the ocular tissues, and do not cause ocular tissue toxicity. It can be used as an ocular absorption enhancer to achieve intraocular drug delivery through a non-invasive route and to increase the ocular bioavailability of the drug. These penetratin derivatives and the ophthalmic drug delivery system thereof are used for eye drop administration, which could replace intraocular injection having poor patients compliance, which greatly enhanced the convenience and safety of the treatment of intraocular and fundus diseases.BACKGROUND OF THE INVENTION[0002]Eye is the most important sensory organ in human body. The unique physiological structure of eye protects it from foreign substances, but it is also disadvantageo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K47/64C07K7/08A61K9/00A61K47/54
CPCA61K9/0048A61K47/545C07K7/08A61K47/549A61K47/64A61K45/00A61K47/42A61K38/10A61P3/00A61P9/10A61P25/00A61P27/02A61P31/02A61P31/04A61P31/10C07K14/43581A61K47/645A61K47/6455A61K47/595A61K47/61
Inventor WEI, GANGJIANG, KUANLU, WEIYUELIU, CHANGTAI, LINGYUGAO, XIN
Owner FUDAN UNIV
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